Clostridium perfringens beta-2 toxin signal sequence, promoter and recombinant prokarote host cells comprising them

ABSTRACT

Recombinant polypeptides are prepared using novel nucleic acids with transcription promoter activity. The recombinant cells containing said nucleic acids are described. A novel method for preparing antigens or antigen fragments, in particular bacterial toxins, preferably  Clostridum  toxins, for preparing immunogenic and/or vaccine compositions is also described.

CROSS-REFERENCE TO A RELATED APPLICATION

The present application is a continuation of PCT/FR98/01999, which wasfiled on Sep. 17, 1998.

The present invention relates to novel nucleic acids with atranscriptional promoter activity. It also relates to a process forpreparing recombinant polypeptides using these nucleic acids, and torecombinant cells containing these nucleic acids. The invention alsorelates to a novel process for preparing antigens or fragments ofantigens, in particular bacterial toxins, more preferably Clostridiumtoxins, for preparing immunogenic and/or vaccine compositions. Stillfurther, it relates to immunogenic and/or vaccine compositions withimproved properties.

More particularly, the present invention relates to the field ofproduction of bacterial toxic proteins, in particular toxins fromClostridium or other pathogenic organisms. In particular, it relates tothe improved production of these toxins, with the aim of producingimmunogenic preparations with an increased vaccinating power.

Diseases of bacterial origin (cholera, dysentery, enteritis, etc) are amajor cause of death in man and in animals. Such diseases areessentially alimentary in origin, linked to the presence of pathogenicbacteria in ingested elements which colonise the mucosal walls thencause toxicity and tissue necrosis. Pathogenic bacteria come fromdifferent genera, particular examples being Actinomycetes, Bacilius,Bordetella, clamydia, Clostridium, Corynebacterium, Escherichia,Fusobacterium, Listeria, Mycobacterium, Mycoplasma, Salmonella,Staphylococcus, Treponemo and Vibro. Of these pathogenic bacteria,Clostridium bacteria form a large class, among which the followingspecies deserve mention: C. absonum, C. baratii, C. bifermentans, C.chauvei, C. difficile, C. ghonii, C. lituseburense, C. novyi, C.perfringens, C. septicum, C. sordellii, C. subterminale and C. tetani.

The pathogenicity of these pathogenic bacteria has been shown to belinked to their production of toxins or enterotoxins. The large majorityof such toxins have now been identified and characterized.

Thus the pathogenicity of strains of Clostridium septicum, known to beresponsible for atraumatic gangrene, is linked to the expression of asingle lethal factor, alpha toxin, the gene of which has been cloned(Ballard et al., Infection and Immunity 63 (1995), 340). The toxinproduced by strains of Clostridium sordellii has been designatedcytotoxin Cs Cyt. Strains of C. perfringens are generally classifiedinto 5 types (A–E) depending on the nature of the toxins they produce(alpha, beta, epsilon, iota and enterotoxin). The toxicity of C. tetaniis linked to the production of a toxin, and Bordetella producespertussis toxin while C. tetani produces tetanus toxin. Other toxinswill be indicated in the text below.

Currently available treatments are essentially prophylactic in nature.Thus vaccine preparations are produced from cultures of pathogenicbacteria strains. The supernatants containing the toxins are thenharvested and undergo different concentration and/or partialpurification steps. The supernatants or their filtrates/concentratesthen undergo an inactivation, step, to produce non virulent toxins, butwhich retain their immunogenic power (toxoids). Different techniques areavailable for inactivating toxins, in particular chemical or genetictechniques which will be described below in detail. Further, prior tothe inactivation step, supernatants from cultures of differentpathogenic organisms are usually combined to obtain cocktails of toxinswith the aim of preparing polyvalent vaccines.

Examples of commercially available vaccines are Miloxan®, sold byRhône-Mérieux (Mérial), France, which protects animals againsttoxi-infections and enterotoxemia due to strains of Clostridiumperfringens and Clostridium sordelli. More particularly, that vaccinepreparation contains toxoids of types B, C and D Clostridiumperfringens, of Clostridium septicum, of Clostridium novyi, ofClostridium tetani, of Clostridium chauvei and of Clostridium sordellii.A further example of a polyvalent vaccine is Gletvax5®, sold byMallinckrodt Veterinary, France, providing protection againstcollibacilosis in young swine and enteritis from type C Clostridiumperfringens. More particularly, that vaccine contains E. coli antigens,as well as toxoids of the beta toxin of type C Clostridium perfringens.

Currently available vaccines, however, have a certain number ofdisadvantages. They are essentially mixtures of culture supernatants,the composition of which is not precisely defined and for which thereproducibility is thus not entirely assured. Further, the production ofpolyvalent vaccines protecting against different pathogenic organismsinvolves separate fermentation of different organisms, meaning that onan industrial scale, culture conditions and safety standards are highlyrestrictive. Further, the efficiency of some vaccines against certaintoxins is limited, in particular those produced in small quantities bypathogenic organisms.

There is thus a genuine need for improving the conditions for preparing,the quality and the efficiency of vaccines against bacterial toxins. Thepresent invention provides an advantageous solution to these problems.

The present invention provides novel nucleic acids enabling expressionof transgenes in bacteria, in particular Clostridium type bacteria. Theinvention also provides constructions enabling larger quantities oftoxins, in particular for vaccine purposes, to be produced in bacteria,in particular in Clostridium type bacteria. In particular, the presentinvention provides strains of recombinant bacteria, in particularClostridium type bacteria, enabling the amplified production of toxins,either Clostridium toxins or toxins from other pathogenic organisms. Theinvention also provides strains of recombinant bacteria, in particularClostridium type bacteria, enabling a number of toxins to besimultaneously produced.

The invention thus describes a process for producing recombinant toxinsenabling the immunogenic character, and thus their protective effect, ofvaccine preparations to be increased.

The invention also describes the production of novel toxins by arecombinant route, enabling the range of existing vaccines to beincreased, in particular as regards the beta 2 toxin of Clostridiumperfringens or other toxins which are produced in small quantities bypathogenic organisms, or which are of low immunogenicity.

The invention also provides a considerably easier industrialimplementation in that the volumes of supernatants produced and thediversity of the productive organisms used can be significantly reduced.

More particularly, in a first aspect, the invention relates to nucleicacids and genetic constructions for improving the production of proteinsin bacteria, in particular bacterial toxins in bacteria of the genusClostridium, in particular Clostridium perfringens.

Thus the Applicant has isolated, from the genome of a type C Clostridiumperfringens strain, the complete gene coding for a toxin, designatedbeta 2 toxin (SEQ ID n^(o) 1). A study of the gene obtained has showedthat the gene also comprises, at the 5′ end, a transcription promotingregion (SEQ ID n^(o) 2), which is effective in bacteria in particular inbacteria from the genus Clostridium, in particular Clostridiumperfringens.

More particularly, in its first aspect, the invention provides a nucleicacid characterized in that it has a transcriptional promoter activityand in that it comprises:

-   (a) all or a portion of sequence SEQ ID NO:3 or a variant thereof;    or-   (b) a sequence hybridizing with all or part of the complementary    strand of sequence SEQ ID NO:3.

More preferably, the nucleic acid of the invention comprises all or aportion of sequence SEQ ID NO:3.

The term “nucleic acid” as used in the invention means any deoxyribosenucleic acid (DNA) or ribonucleic acid (RNA). More particularly, the DNAcan be a complementary DNA (cDNA), a genomic DNA (gDNA) or a syntheticDNA. In the present invention, the term “nucleic acid” is alsosynonymous with “polynucleotide”. The nucleic acids of the invention canbe of a variety of origins, in particular bacterial, synthetic orsemi-synthetic. They may be isolated using any known molecularbiological technique, using structural data and sequences provided inthe present application. Thus these nucleic acids can be isolated fromlibraries using hybridisation techniques. They can also be synthesizedchemically or genetically.

The term “portion” or “fragment” of nucleic acid means any nucleic acidcomprising at least a portion of the sequence under consideration (forexample sequence SEQ ID NO:3 and which retains a transcriptionalpromoter activity. The sequence portion advantageously contains at least50 bp, more preferably at least 100 bp. These “portions” can readily begenerated using conventional molecular biological techniques, either byenzymatic cleavage and digestion from the fragments describes, or bysynthesis using nucleic acid synthesisers.

The term “hybridising” as used in the present invention means anyhybridisation under normal conditions, which may be stringent or nonstringent, as defined below. An example of stringent hybridisationconditions is: Hybridisation at 42° C., 50% formamide, 5×SSC, 1×Denardt;Wash at 65° C. in 0.1×SSC, 0.1% SDS. Non stringent conditions are:Hybridisation at 37° C., 40% formamide, 5×SSC, 1×Denardt; Wash at 50° C.in 1×SSC, 0.1% SDS. Stringent conditions are particularly suitable whenthe nucleic acids are present in small quantities and/or are in purifiedform. Non stringent conditions are more suitable when the nucleic acidpresent in large quantities and are significantly represented in thesample. Advantageously, “hybridising” sequences are sequences whichhybridize under stringent conditions, and which thus have a high degreeof structural homology with the sequence under consideration (forexample SEQ ID NO:2) or its fragments. Further, hybridising sequencescan include a region enabling hybridisation and a contiguous regionwhich is not hybridising, but corresponding to flanking regions.

In addition, the transcriptional promoter activity of the “fragments” or“portions” and “hybridising sequences” can readily be determined by theskilled person using the methodology described in the examples. Inparticular, the activity of the fragments/hybridising sequences can beverified by introducing these nucleic acids to the 5′ end of a markergene, then studying the expression of that marker in a population ofcells such as bacteria from the genus Clostridium, in particularClostridium perfringens.

The examples below show that the nucleic acids of the invention cansignificantly amplify the expression of a protein in a bacterium, inparticular in a Clostridium perfringens.

Thus for a wild strain producing beta 2 toxin, the production level isincreased by a factor of about 40 to 80 in the presence of a nucleicacid of the invention. Further, the following examples show that thesenucleic acids enable the expression of significant levels ofheterologous proteins in bacteria from the genus Clostridium. Inparticular, the examples show that the nucleic acids of the inventioncan produce heterologous toxins in these bacteria.

In this respect, the invention also concerns a cassette for expressionof a transgene, characterized in that it comprises, in the 5′→3′direction:

-   a nucleic acid as defined above; and-   said transgene.

In the cassette of the invention, the nucleic acid and the transgene areoperationally connected together (i.e., so that the nucleic acid enablesexpression of said transgene).

Advantageously, the cassette of the invention further comprises atranscriptional terminator, at the 3′ end of the transgene.

Further, the cassette of the invention can also advantageously comprisea secretion signal which can induce or increase secretion of theexpression product of the transgene by the cells. Advantageously, thissecretion signal is located between the nucleic acid of the inventionand the transgene, in the same reading frame as the latter.

To this end, the inventors have also demonstrated the existence in theidentified gene of such a secretion signal, which is particularly activein bacteria from the genus Clostridium. This signal is represented byresidues 268–357 in sequence SEQ ID NO:1, and separately in SEQ ID NO:4.This signal, or any variant or active fragment thereof, constitutes anadvantageous embodiment of the invention.

As indicated above, the invention is particularly suitable for theproduction of toxins or fragments or variants of toxins. Thus in aparticular embodiment, the expression cassette of the invention ischaracterized in that the transgene codes for a toxin or a toxinfragment or variant. More particularly still, the transgene codes for atoxin or a toxin fragment or variant of a pathogenic bacterium.

The term “toxin” as used in the invention means any peptide, polypeptideor protein produced by a pathogenic bacteria, and involved in saidpathogenic activity. It may be a factor which is directly responsiblefor the toxicity of the bacterium, or it may participate in thattoxicity. A toxin “fragment” can be constituted by any portion of atoxin, which has retained certain immunogenic features of the toxin. Inparticular, bacterial toxins have been described as often presentingdifferent distinct functional domains, in particular a domain involvedin toxic activity (catalytic site) distinct from other domains involvedin site recognition or in interactions with partners. A toxin “fragment”of the invention is advantageously constituted by a domain which isdeprived of toxic activity, but which retains an immunogenic power. Atoxin variant may, for example, be constituted by a derivative resultingfrom genetic modifications of the sequence coding for said toxin ortoxin fragment. Examples of such genetic modifications are mutations,deletions, fusions, etc.. In general, mutations affect 1 to 10 residues,preferably 1 to 5 residues. These mutations are mutations which modifythe amino acid coded for, and thus which modify the protein sequence.Deletions can be internal or terminal deletions. They can affect up to40% of the entire sequence. Fusions consist of introducing supplementalregions at the 5′ and/or 3′ end of the sequence, or possibly ofinserting such regions into the sequence. These modifications can becarried out with the aim either of reducing or of removing the toxicityof these proteins, or of improving their production or stability, forexample. Such genetic modifications can be carried out underconventional molecular biological conditions, and are illustrated in theprior art and in the remainder of the text of the present application.

More particularly, the cassettes of the invention are suitable for theproduction of the following toxins or variants:

-   The beta 2 toxin of Clostridium perfringens or any immunogenic    fragment. The hydrophilicity profile of beta 2 toxin is shown in    FIG. 8. This profile shows a number of hydrophilic regions, which    define particular fragments within the context of the invention.    These regions are in particular located at amino acid residues    40–55, 105–120, 160–170, 175–188, 200–210 and 250–260 as shown in    SEQ ID n^(o) 1. Further, fragments of the beta 2 toxin which are    free of toxicity are trypsic fragments of 24, 15 and 13 kDa    described in the examples. Constructions for expressing this toxin    are described in the examples.-   The beta 1 toxin of Clostridium perfringens or any immunogenic    fragment. The sequence for this toxin has been described in the    literature (Hunter et al., Infect. Immun. 61 (1993), 3958–3965).    Constructions for expressing this toxin are described in the    examples.-   The iota toxins of Clostridium perfringens or any immunogenic    fragment. The sequence for genes coding for iota 1 toxins (gene 1a)    and iota 2 toxins (gene 1b) have been described in the literature    (Perelle et al., Infect. Immun. 61 (1993) 5147–5156).-   The alpha toxin of C. novyi or any immunogenic fragment. The gene    sequence for this toxin (tcnα gene) has been described in the    literature (Hofmann et al., Mol. Gen. Genet. 247 (1995) 670–679).-   The alpha toxin of C. septicum or any immunogenic fragment. The gene    sequence for this toxin has been described in the literature    (Ballard et al., Infect. Immun. 63 (1995) 340–344).-   The A and B toxins of C. difficile or any immunogenic fragment. The    gene sequence for this toxin has been described in the literature,    as well as different immunogenic regions (Von Eichel-Streiber et    al., Mol. Gen. Genet. 233 (1992) 260–268; Von Eichel-Streiber et    al., J. Gen. Microbio. 135 (1989) 55–64; Von Eichel-Streiber et    al., J. Bacteriol. 174 (1992) 6707–6710).-   The epsilon toxin of C. perfringens (Worthington et. al.,    Onderstepoort J. Vet. Res. 40 (4) (1973) 145–152/Hunter et al.,    Infect. Immun. 60, (1992) 102–110).-   The enterotoxin of C. perfringens (McClane, Toxicon. 34 (1996)    1335–1343).-   The toxin of C. chauvoei (Crichton et al., Australian Vet. J.    63 (1986) 68).-   The L cytotoxin of C sordellii or any immunogenic fragment. The gene    sequence for this toxin has been described in the literature (Green    et al., Gene 161 (1995) 57–61), also different immunogenic regions.    In particular, this toxin is constituted by a protein of about 270    kDa and different antigenic fragments have been described.-   The pertussis toxin.-   The tetanus toxin, the botulism toxin, in particular the C fragment    of these toxins which is the immunogenic fragment (Makoff et al.,    Bio/Technology 7 (1989) 1043; Figueiredo et al., Infect. Immun.    63 (1995) 3218–3221; Wells et al., Molecular Microbiol. 8 (1993)    1155–1162; Boucher et al., Infect. Immmun. 62 (1994) 449–456; Clare    et al., Bio/Technology 9 (1991) 455; Clayton et al., Infect.    Immunol. 63 (1995) 2738–2742).

The nucleic acids and/or expression cassettes of the invention can beinserted in a vector which constitutes a further aspect of the presentinvention. It is advantageously a vector which is functional inbacteria, i.e., capable of penetrating into bacteria and transportingthe nucleic acids of the invention thereto. More preferably, such avector comprises either a functional origin of replication in abacterium, or sequences enabling it to integrate into the genome of abacterium. More particularly, it is a plasmid, phage, episome, etc.Further, certain vectors may advantageously comprise two origins ofreplication, one functional in bacteria of the genus E. coli, and theother functional in bacteria of the Clostridium type, for example.

The vector of the invention is particularly preferably a vector which isfunctional in bacteria from the genus Clostridium, in particular inClostridium perfringens bacteria. As an example, it may be a vectorderived from the plasmid pAT19 described by Trieu-Cuot et al. (Gene 102(1991) 99–104). Such a derivative is, for example, the vector pMRP353 orthe vector pMRP268 as shown in FIGS. 2–4.

The invention further concerns any recombinant cell comprising a nucleicacid or an expression cassette or a vector as defined above. Therecombinant cell is advantageously a prokaryote cell, preferably abacterium. Particularly advantageously, the cell of the invention is abacterium of the genus Clostridium selected from C. absonum, C. baratii,C. bifermentans, C. chauvei, C. difficile, C. ghonii, C. lituseburense,C. novyi, C. perfringens, C. septicum, C. sordellii, C. subterminale andC. tetani. More preferably still, it is a C. perfringens bacterium.

The recombinant cells of the invention can be prepared using any of thetechniques known to the skilled person for introducing a nucleic acidinto a cell. It may be a physical technique (electroporation,bombardment, gene gun, etc.), a chemical technique (precipitation withCaPO₃, use of chemical transfer agents: cationic lipids, polymers, etc.,or other methods such as cell fusion, conjugation, etc. (Scott et al.,Gene 82 (1989) 327–333; Phillips-Jones, FEMS Microbiol. Letters 66(1990) 221–226; Allen et al., FEMS Microbiol. Letters 70 (1990)217–220).

Further, a recombinant cell of the invention may comprise a plurality ofcassettes or vectors of the invention comprising different transgenes,and thus produce either different toxins, or different fragments of thesame toxin.

The invention also relates to a process for producing a polypeptidecomprising introducing a transgene coding for said polypeptide into ahost cell under the control of a promoter as defined in the invention,then recovering said polypeptide.

A particular process for producing polypeptides of the inventioncomprises culturing a recombinant cell as defined above comprising anexpression cassette or a vector, the transgene coding for saidpolypeptide.

More particularly, in the process of the invention, the cell is abacterium of the genus Clostridium, more preferably C. perfringens.

The process of the invention is particularly suitable for the productionof a toxin or a toxoid. The term “toxoid” is well known to the skilledperson, and designates any inactivated form of a toxin, i.e., deprivedof toxic nature, but retaining the immunological properties of thetoxin. More preferably, the process of the invention is used to producea toxin (or a corresponding toxoid) selected from the group comprisingthe alpha, beta (beta 1 and beta 2), iota (1 and 2), epsilon andenterotoxin of C. perfringens, pertussis toxin, tetanus toxin, the alphatoxin of C. septicum, the alpha toxin of C. noyvi, the A and B toxins ofC. difficile or the L cytotoxin of C. sordellii.

More generally, then, the invention concerns the use of a nucleic acidas defined above for the production of polypeptides.

The invention also concerns a process for preparing an immunogeniccomposition comprising the following steps:

-   a) expressing one or more toxins (or the corresponding toxoids) in a    cell, as defined above;-   b) harvesting the supernatant;-   c) optionally, treating the supernatant to purify or concentrate the    toxin(s) or toxoid(s);-   d) inactivating the toxin(s); and-   e) optionally, packaging the inactivated toxin(s) or the toxoid(s).

In the process of the invention, an optional supplementary step carriedout before or after step d) comprises grouping the supernatant withother culture supernatant(s) containing a different or identical toxin,or a corresponding toxoid. These other supernatant or supernatants mayoriginate from recombinant strains as defined in the invention, or fromany other strain, which may or may not be recombinant, producing thetoxin under consideration. In the process of the invention, twosupernatants are advantageously combined, which originate from culturesof recombinant bacteria of the invention producing a different toxin.

A) PRODUCTION OF SUPERNATANTS

The first step in producing the immunogenic compositions of theinvention consists of producing culture supernatants containing thetoxin or toxins under consideration. At least a portion of thesupernatants advantageously originate from a recombinant bacterialstrain comprising a cassette or a vector as defined above. Forpolyvalent vaccines, a plurality or all of the supernatants can besupernatants from recombinant bacterial strains comprising a cassette ora vector as defined above. More preferably, they are recombinant strainsof Clostridium, more preferably C. perfringens. One advantage of theprocess of the invention is that it limits the number of differentorganisms used for fermentation. Further, the recombinant strains usedcan also comprise a plurality of cassettes or vectors of the invention,such that they simultaneously produce a plurality of toxins. Thisembodiment advantageously further reduces the number of fermentations inthe production process.

The production can be carried out under the culture conditions describedabove, in fermenters of 50 to 1500 litres. When the supernatantscontaining the toxins have been produced, they undergo differenttreatments.

B) HARVESTING SUPERNATANTS

The supernatants are harvested using conventional biological techniqueswhich are well known to the skilled person. In particular, thesupernatants can be recovered by simple filtration to separate thecells.

C) TREATMENT OF SUPERNATANTS

In order to improve the quality of the final preparation, thesupernatants can optionally undergo supplementary treatments such asfiltration, centrifugation, concentration, etc. These treatments clarifythe supernatants, and partially purify the toxins. These techniques areknown to the skilled person.

Further, in the case of polyvalent vaccines, different supernatants canbe collected at this stage.

D) INACTIVATION OF TOXINS

In order to prepare an effective immunogen, it is of course importantthat the antigen used is free of the toxicity of the toxin against whichprotection is sought. With the aim of generating these inactivatedtoxins (toxoids), different techniques are possible (Rappuoli et al.,Int. Arch. Allergy Immunol. 108 (1995) 327–333).

-   Chemical Inactivation    -   Chemical toxin inactivation has been long described, and        continues to be used for numerous immunogenic preparations. Thus        with the aim of inactivating bacterial toxins without affecting        their immunogenic properties to too great an extent, the        supernatants of the process of the invention can be treated with        the following compounds: formol, β-propiolactone, iodine,        formaldehyde or glutaraldehyde. More precise conditions and        doses which can be used are known to the skilled person, and        have been described, for example, by Rappuoli R (In Woodrow G C,        Levine M M (eds): New Generation Vaccines, New York, Dekker,        1990 p. 251–268), hereby incorporated by reference.-   Physical Inactivation    -   The toxins can also be physically inactivated, for example by        irradiation.-   High Pressure Inactivation    -   High pressure treatment can also be used to effect inactivation.-   Genetic Inactivation    -   A further approach to inactivating toxins resides in genetic        modification of their primary structure. In this case, the        products liberated into the supernatant are already toxoids, and        it is not necessary to carry out a supplementary inactivation        step. Genetic inactivation (or detoxification) essentially        consists of modifying the nucleic acids coding for the toxins,        such that one or more amino acids are changed and the protein        produced is free of toxicity. As an example, it is possible to        replace the amino acids involved in enzymatic activity, and thus        toxicity, with different amino acids which do not have this        activity. It is also possible to delete regions of the toxin so        as to produce non-toxic, immunogenic fragments. This strategy        can be carried out when epitopes of the toxins have been        identified (for example by epitope scanning) or are identifiable        (for example from a hydrophobicity profile). This strategy can        also be applied to the production of fragments (for example        trypsic fragments) for which the absence of toxicity has been        demonstrated. Further, genetic modification can also be carried        out by random mutagenesis and selecting clones producing a        toxoid. Such a strategy has already been successfully carried        out to produce toxoids of the diphtheria toxin by mutagenesis        using nitrosoguanidine (NTG) (Giannini et al., Nucleic Acids        Res. 12 (1984) 4063–4069). Further, the production of toxoids by        site-specific mutagenesis has also been successfully carried out        using genes of pertussis and cholera toxins (Pizza et al.,        Science 246 (1989) 497–500; Pizza et al., J. Exp. Med. 6 (1994)        2147–2153; Fontana et al., Infect. Immun. 63 (1995) 2356–2360).    -   The toxoids produced are then directly used to prepare vaccine        compositions.

E) FORMULATION

The toxoids are generally packaged using conventional pharmocologicaltechniques in a manner suitable for vaccine use. The toxoids arepreferably formulated in the presence of adjuvants or excipients whichare suitable for the production of injectable solutions. In particular,injection is advantageously carried out subcutaneously or systemically.The doses of antigen (toxoid) used are generally those which will ensurethe best protection without inducing a significant secondary reaction.The conditions and sites for injection, and techniques for determiningdoses, are illustrated in detail in the pharmacopia (Vaccinum ClostridiiPerfringentis, Chap. 363).

The invention also relates to any immunogenic preparation comprising atoxin produced in a recombinant strain as defined above. Moreparticularly, the invention relates to any immunogenic preparationcomprising a toxoid of recombinant beta 2 toxin, optionally combinedwith other toxoids.

The present invention will now be described in more detail with the aidof the following examples which should be considered to be illustrativein nature and are in no way limiting.

DESCRIPTION OF THE FIGURES

FIG. 1: Cloning strategy for the complete gene for the beta 2 toxin ofC. perfringens. H: HindIII; S: Sau3A

FIG. 2: Schematic representation of vector pMRP268.

FIG. 3: Schematic representation of vector pMRP353.

FIG. 4: Schematic representation of a plasmid carrying the beta 2promoter.

FIG. 5: SDS-PAGE and Western Blot analysis of purified recombinant beta2 toxin: (A) SDS gel (0.1%)-PAGE (10%) stained with Coomassie blue forthe beta 2 recombinant toxin (4.5 μg); (B) Corresponding Western Blotobtained with anti-beta 2 antibodies.

FIG. 6: Toxicity study of purified beta 2 toxin on 1407 cells. Photos of1407 cells taken using a phase contrast microscope, control (A); treatedwith 20 μg/ml of beta 2 toxin for 18 hours (C) (B) and (D): view ofactin cytoskeleton.

FIG. 7: Sensitivity of beta 2 toxin to trypsine. The beta 2 toxin (165μg/ml) was incubated without (track 7) or with 16 ng/ml (track 1), 160ng/ml (track 2), 400 ng/ml (track 3), 1.6 μg/ml (track 4) 4 μg/ml (track5) and 16 μg/ml (track 6) of trypsin.

FIGS. 8A and 8B: Hydrophobicity profile for beta2 toxin (SEQ ID NO:2).

SEQ ID NO:1: Gene sequence for beta 2 toxin of type C Clostridiumperfringens (nucleotides 1–327 of SEQ ID NO:1 is shown as SEQ ID NO:8).

SEQ ID NO:3: Sequence for beta 2 toxin gene promoter of type C.Clostridium perfringens.

SEQ ID NO:4: Sequence for secretion signal of beta 2 toxin gene of typeC Clostridium perfringens.

SEQ ID NO:6: Sequence of primer P318.

SEQ ID NO:7: Sequence of primer P292.

MATERIALS AND METHODS 1. Bacterial Strains and DNAs Used

The bacterial strains used are mentioned in Tables 1 and 2. Clostridiumstrains were cultivated in the presence of Trypticase (30 g/litre),yeast extract (20 g/litre), glucose (5 g/litre) and cysteine-HCl (0.5g/litre), pH 7.2 (TGY medium) at 37° C. under anaerobic conditions.

Total DNA and plasmidic DNA from Clostridium were extracted and purifiedusing the technique described by Perelle et al. (Infect. Immun. 61(1993) 5147–5156).

Plasmids pUC19 and pUC18 (Appligene Strasbourg, France) were used forthe cloning experiments in TG1 E. coli, and shuttle vectors pAT19(Trieu-Cuot et al., Gene 102 (1991) 99–104) and pJIR750 (Bannan andRood, Plasmid 229 (1993) 233–235) were used for cloning and expressionexperiments in Clostridium, in particular in Clostridium perfringens667–76, a negative lecithin strain.

-   2. The synthetic oligonucleotides and hybridisation experiments were    those described by Perelle et al., 1993 (supra).-   3. The PCR amplification experiments were carried out in a total    volume of 100 μl using 100 ng of DNA as described above (Perelle et    al, 1993, supra).-   4. Ligations and transformations were carried out using standard    molecular biological protocols (Sambrook et al., Molecular Cloning:    A Laboratory Manual, Cold spring Harbor Laboratory Press, Cold    Spring Harbor, N.Y., 1989).-   5. The beta 2 toxin was purified and microsequenced using the    protocol described in International patent application WO 95/17521.-   6. The cytotoxicity was determined using the following test:    intestinal 1407 cells (ATCC) were cultivated in modified Dulbecco    medium (DMEM) supplemented with 5% of foetal calf serum. The 1407    cells were spread onto 96 well culture plates (Falcon, Becton    Dickinson) and cultivated for 24 hours at 37° C. in an incubator    containing 5% CO₂ to obtain monolayers. Series dilutions by a factor    of 2 of samples with a final volume of 100 μl wee added to the    monolayers. The cells were examined after 18 hours incubation to    detect any change in morphology. The actin cytoskeleton was observed    by immunofluorescence using fluorescent phalloidine isothyocyanate    (1 μg/ml, Sigma) using the technique described by Giry et al.    (Infect. Immun. 63 (1995) 4065–4071).

EXAMPLES A—Cloning of the Complete Beta 2 Toxin of Type C ClostridiumPerfringens

This example describes the cloning of the complete gene of the beta 2toxin, i.e., including the regulation and addressing signals at the 5′end.

A 676 bp fragment essentially comprising the region coding for themature form of the protein was isolated by amplification using the P279and P280 deduced by microsequencing the protein (WO 95/17521). Thisfragment did not include the complete gene, in that no regulation signalwas present at the 5′ end of the gene. Further, the existence ofaddressing sequences was not revealed nor was it deducible from thisfragment. With the aim of cloning the complete gene, the inventorsfirstly used this fragment as a probe to isolate, by hybridisation froma gene library, a fragment with a larger size comprising the 5′ region.However, none of these experiments either detected or obtained acorresponding fragment, whatever the hybridisation conditions used. Theinventors thus designed a different strategy to attempt to isolate afragment carrying the 5′ regions of the gene. In this regard, theinventors firstly circularised the matrix for amplification, then usedinverse PCR on the DNA thus obtained using the P292 and P318 primers thesequence of which is as follows:

P318: 5′-GAAATGTTTACAACTGTATTAATATCGTAG-3′ (SEQ ID NO:6)

P292: 5′-TCAAGTTTGTACATGGGATGATG-3′ (SEQ ID NO:7)

The location of these primers on the gene is indicated in sequence SEQID n^(o) 1. The cloning strategy is shown in FIG. 1. The amplifiedfragment thus obtained was then sub-cloned in the pUC18 plasmid cleavedwith SmaI, to generate the pMRP224 vector (FIG. 1).

The complete 1392 bp sequence of the fragment obtained is shown insequence SEQ ID n^(o) 1.

B—Identification of Promoter Regions

The sequence obtained in Example A (SEQ ID NO:1) comprises an openreading frame coding for the mature beta 2 toxin (residues 358 to 1122),and regulating or addressing regions located at the 5′ and 3′ end. Thepromoter region in the gene of the beta 2 toxin of type C Clostridiumperfringens can be pinpointed on this sequence as comprising residues 1to 267. This promoter sequence in the beta 2 toxin gene of type CClostridium perfringens is shown separately in sequence SEQ ID NO:3.This sequence includes a consensus ribosome binding site (GGGGGG)located 7 nucleotides upstream of the start codon ATG, i.e., atpositions 255–260 in sequence SEQ ID NO:3.

This region, or any fragment or variation thereof, can be isolated fromsamples of Clostridium nucleic acids using suitable probes (for examplecorresponding to sequence SEQ ID NO:3 or a fragment thereof) or bychemical synthesis, or by enzymatic digestion from the plasmids of theinvention, in particular the plasmid pMRP268, deposited on 8^(th) Aug.1997 at the Collection of the Institut Pasteur (CNCM: CollectionNationale de Cultures de Microorganismes), accession number I-1911.

The activity of the transcriptional promoter in this region or fragmentsor variants can be determined in different manners, in particular byinserting this region upstream of a reporter gene, and verifying thepresence of the transcription or translation product of the reportergene in a suitable cell host, in particular a bacterium from the genusClostridium, more preferably C. perfringens.

The reporter gene can, for example, be the LacZ gene or the gene codingfor luciferase.

The construction of these expression cassettes or vectors is shown inExamples D onwards, along with the transformation conditions fordifferent cell hosts.

C—Identification of Addressing Regions

In addition to an open reading frame and transcription regulationregions (Example B), the sequence obtained in Example A (SEQ ID NO:1)also comprises addressing signals enabling a protein or peptide to bedirected during synthesis towards the host cell secretion routes. Theaddressing region (secretion signal peptide) of the beta 2 toxin gene oftype C Clostridium perfringens can be seen in sequence SEQ ID NO:1 asincluding residues 268 to 357. This signal peptide sequence in sequenceSEQ ID NO:4. This region codes for 30 amino acids, comprising ahydrophobic region (residues 6–26), probably forming a transmembranedomain, bordered by charged amino acids (Lys2, Lys3, Lys7 and Lys27).Further, the junction region between this signal sequence and the matureprotein (Ala30—Lys31) corresponds to the (Ala—X) cleavage site of themajor portion of bacterial signal peptidases.

This region, or any fragment or variant thereof, can be isolated fromsamples of Clostridium nucleic acids using suitable probes (for examplecorresponding to sequence SEQ ID NO:4 or a fragment thereof) or bychemical synthesis, or by enzymatic digestion from plasmids of theinvention, in particular plasmid pMRP268, deposited on 8^(th) Aug. 1997in the Institut Pasteur collection (CNCM), accession number I-1911.

The signal sequence activity in this region or of fragments or variantscan be checked in different manners, in particular by inserting thisregion upstream of a reporter gene, and verifying the presence of thetranslation product of this reporter gene in the culture supernatant ofa suitable host cell, in particular a bacterium from the genusClostridium, more preferably C. perfringens.

The construction of expression cassettes or vectors comprising this typeof secretion signal is illustrated in the following examples which alsogive the transformation conditions in different cell hosts.

D—Construction of Expression Cassettes and Vectors

The regulation and addressing regions described in the invention can beinserted into any conventional expression vector, or used to constructexpression cassettes.

These cassettes and vectors are particularly suitable for expression(and optionally secretion) of recombinant proteins in bacteria from thegenus Clostridium, in particular Clostridium perfringens. It should beunderstood that any cell type in which these regions are functional canbe used. These regions are particularly advantageous for expression ofbacterial toxins, in particular toxins of bacteria from the genusClostridium. The construction of suitable cassettes and vectors isdescribed below.

D1. Construction of vector pMRP268 (FIG. 2)

The vector pMRP268 carries the following elements:

-   an origin of replication OriR enabling its replication in an E. coli    bacterium;-   an origin of replication OriT enabling its replication in a    bacterium from the genus Clostridium;-   two marker genes (orfE and erm) enabling transformants in E. coli    and Clostridium to be selected;-   an expression cassette comprising, in the 5′→3′ direction, the    promoter for the beta 2 toxin of Clostridium perfringens, the    secretion signal for the beta 2 toxin of Clostridium perfringens,    and the sequence coding for the beta 2 toxin of Clostridium    perfringens.

This vector was constructed from plasmid pAT19, by introducing theexpression cassette into the cloning multi-site. More particularly, thecassette was obtained by amplification in the beta 2 gene of Clostridiumperfringens of sequence SEQ ID n^(o) 1 using primers P385 and P393, thesequence and position of which are shown in SEQ ID n^(o) 1, using VentPolymerase (Biolabs) following the recommendations of the manufacturer.The resulting amplification product was inserted into the EcoRI—PstIsites of plasmid pAT19. C. perfringens 667–76 strain not expressinglecithin and containing plasmid pMRP268 was deposited at the CNCM,Institut Pasteur, on 8^(th) Aug. 1997, accession number I-1911.

D2. Construction of vector pMRP353 (FIG. 3).

The vector pMRP353 carries the following elements:

-   an origin of replication OriR enabling its replication in an E. coli    bacterium;-   an origin of replication OriT enabling its replication in a    bacterium from the genus Clostridium;-   two marker genes (orfE and erm) enabling transformants in E. coli    and Clostridium to be selected;-   an expression cassette comprising, in the 5′→3′ direction, the    promoter for the beta 2 toxin of Clostridium perfringens, the    secretion signal for the beta 2 toxin of Clostridium perfringens,    and the sequence coding for the beta 1 toxin of Clostridium    perfringens.

This vector was constructed from plasmid pMRP268 by substituting thesequence coding for the beta 2 toxin of Clostridium perfringens by thatcoding for the beta 1 toxin. The cDNA coding for the beta 1 toxin wasobtained by amplification from strain NTCT8533 (Table 1) using primersintroducing an NcoI site at the 5′ end (P321) and a PstI site at the 3′end (P322).

D3. Construction of a vector for expression of any cDNA

It is clear that the vectors described in D1 and D2 above can be used toexpress any cDNA of interest under the control of a promoter region fromthe beta 2 gene, by substitution of the coding sequence, as illustratedin Example D2. Further, equivalent vectors can be constructed from otherconventional plasmidic skeletons carrying other origins of replicationand selection markers.

FIG. 4 represents a vector carrying the promoter for the beta 2 gene ofClostridium perfringens, followed by a cloning multi-site enabling anycDNA of interest to be introduced.

E—Production and Purification of Recombinant Beta 2 Protein inClostridium

Plasmid pMRP268 was introduced into the C. perfringens 667–76 strain byelectroporation (Perelle et al., 1993). The recombinant strains wascultivated overnight in a TGY medium containing 30 μg/ml oferythromycin, under anaerobic conditions at 37° C. The culturesupernatant was then precipitated by saturation in the presence of 60%ammonium sulphate. The precipitate was dialysed against 10 mM Tris-HCl,pH 7.5, and charged onto a column of DEAE Sepharose CL6B. The column wasthen washed and eluted in the presence of 0.1 M NaCl in the same buffer.The eluted material was dialysed against 10 mM PIPES-HCl, pH 6.5, andcharged onto a new column of DEAE Sepharose CL6B equilibrated with thesame buffer. After washing, the column was eluted with a 0–0.1 Mgradient of NaCl in a PIPES buffer. The fractions containing purifiedrecombinant beta 2 toxin were concentrated. The apparent molecularweight of the recombinant protein, determined by SDS-PAGE, was 28 kDa,which agreed with the molecular weight calculated from the sequence(FIG. 5).

The results obtained thus show:

-   that it is possible to produce and secrete a recombinant toxin in    Clostridium under the control of the beta 2 promoter;-   that the recombinant toxin can be purified;-   that the structure of the recombinant toxin does not appear to    alter;-   that the levels of expression obtained are higher by a factor of 10    than those observed in a wild strain of Clostridium.

One experiment was carried out under similar conditions, but introducingthe expression vector not into the 667–76 strain (which does not producethe beta 2 toxin) but into a wild Clostridium strain. The resultsobtained show that the presence of the vectors of the invention in theClostridium strains could increase the levels of production of the beta2 toxin by a factor of 40 to 80.

These results demonstrate the advantages of the present invention. Thusthe possibility of producing high levels of different types of toxins ina Clostridium strain can improve the immunogenic power of thesesupernatants, broaden the range of vaccines to encompass toxins whichare naturally produced in only small amounts, and simplify theindustrial production of vaccine compositions.

F—Production of the Recombinant Beta 1 Protein in Clostridium

Plasmid pMRP353 was introduced into the C. perfringens 667–76 strain byelectroporation (Perelle et al., 1993). The recombinant strain wascultivated overnight in a TGY medium containing 30 μg/ml of erythromycinunder anaerobic conditions at 37° C. The culture supernatant was treatedas in Example E.

This experiment demonstrated the presence of recombinant beta 1 in thesupernatants.

This example thus illustrates the capacity of the constructions of theinvention to produce and secrete heterologous toxins (i.e., differentfrom beta 2 or originating from pathogenic organisms other than C.perfringens) in Clostridium strains.

G—Production of Immunogenic Compositions

As indicated above, the present invention now enables before largequantities of different types of toxins to be produced in a strain ofClostridium which can form part of vaccine compositions. The inventionthus improves the immunogenic power of these vaccines, broadens therange of vaccines to encompass toxins which are only naturally producedin small amounts, and simplifies the industrial production of vaccinecompositions.

In particular, the present invention enables vaccines containing atoxoid of the beat 2 toxin to be produced, i.e., an inactivated form, toinduce improved protection against infections by Clostridium. Theadvantages of such compositions are illustrated by the demonstration ofimportant toxic properties of the beta 2 toxin.

G1. Properties of the Purified Recombinant Beta 2 Toxin

Purified beta 2 toxin was intravenously injected into mice. The resultsobtained showed that this toxin was lethal for mice in doses of lessthan 3 μg. Further, the results shown in FIG. 6 show that the beta 2toxin was also toxic for 1407 cells.

Further, the effect of treating beta 2 with trypsine on the activity ofthis toxin was evaluated. As shown n FIG. 7, 16 ng/ml of trypsinecleaved the beta 2 toxin into a 24 kDa constituent and at higherconcentrations into two 13 and 15 kDa peptides. Cytotoxicity testscarried out with these trypsic digestion products showed a total absenceof toxicity. Thus trypsin induces a loss of toxicity of the beta 2toxin, and the peptides generated can be used as toxoids.

In order to determine the importance of the beta 2 toxin in thepathogenicity of Clostridium, the presence of the corresponding gene wasanalysed in 57 different strains of Clostridium (Tables 1 and 2). Theresults obtained shows that certain strains of type B and C Clostridiumcarry the beta 2 gene. In addition, of 27 strains isolated from youngswine presenting with necrotic enteritis type lesions, 44% carried thebeta 2 gene. Further, only the beta 2 gene was detected in all strainsisolated from horses dying from colitis symptoms and in whichClostridium perfringens was harvested in large quantities (over 10⁶/g)from intestinal extracts. These results show the correlation between thebeta 2 toxin and certain animal diseases, and the importance of beingable to generate vaccine compositions wherein one of the antigens is atoxoid of the beta 2 toxin, in particular to vaccinate young swine andhorses.

G2. Production of Immunogenic Compositions

This example illustrates the production of immunogenic compositions orvaccine compositions for protecting the organisms concerned againstinfections by pathogenic bacterial strains.

a) Polyvalent or monovalent compositions.

As indicated above, vaccine compositions can be monovalent (directedagainst a single toxin) or polyvalent (directed against a plurality oftoxins). Commercially available toxins are generally polyvalent(Miloxan, Gletvax5). Preferred immunogenic compositions of the inventionare also polyvalent. The immunogenic compositions of the inventionadvantageously comprise at least one recombinant toxoid produced in arecombinant cell of the invention. A further preferred immunogeniccomposition in the context of the invention advantageously comprises atoxoid of the beta 2 toxin of C. perfringens. The immunogeniccompositions of the invention can also comprise any toxin mentionedabove.

b) Production of an immunogenic composition against the beta 2 toxin

In order to prepare such a composition, the culture supernatant from theC. perfringens strain transformed by the vector pMRP268 (Example E) washarvested using conventional techniques. This supernatant wascentrifuged, then filtered and concentrated to obtain a preparationwhich was enriched in recombinant beta toxins. This preparation was thentreated with formol to inactivate the toxins present. The treatmentefficiency was determined by incubating 1407 cells in the presence of asample of this preparation.

This preparation was then used as an immunogen to induce the productionof antibodies in an organism. The capacity of the antibodies produced toinhibit an infection by pathogenic strains of Clostridium could then bedetermined as described in the pharmacopia (Vaccinum Clostridiumperfringens).

c) Production of a polyvalent immunogenic composition

In order to prepare such a composition, the enriched compositionobtained in Example b) above was mixed, before or after inactivation,with one or more other culture supernatants or derivative preparationscomprising a toxin or the corresponding toxoid, such as a pertussis,cholera and/or tetanus toxoid. The resulting preparation was thenchecked and used as described in Example b).

TABLE 1 Strains of type C C. perfringens Presence of beta 2 toxin geneNCTC8533 − NCTC6121 − ATCC3628 − NCTC8081 − NCTC3180 + NCTC3182 +

TABLE 2 Presence of genes Total cpb2+, cpb2+, cpb2−, Isolates fromClostridium cpb1− cpb1+ cpb1− Young swine 27 12 12 1 Horses 15 16 0 0Foodstuffs 15 2 0 0 cpb2: gene of beta 2 toxin; cpb1: gene of beta 1toxin

1. A purified nucleic acid comprising: (a) SEQ ID NO:3; or (b) asequence from a Clostridium strain hybridising over the full length ofthe complementary strand of SEQ ID NO:3 under stringent conditions,which comprise hybridising at 42° C. in 50% formamide at 5×SSC and1×Denhardt's; wherein said purified nucleic acid has transcriptionalpromoter activity.
 2. The purified nucleic acid according to claim 1,which comprises SEQ ID NO:3.
 3. The purified nucleic acid according toclaim 1, which is a Clostridium perfringens beta 2 toxin promoter.
 4. Anexpression cassette comprising, in the 5′ to 3′ direction, the purifiednucleic acid according to claim 1 and a transgene to be expressed. 5.The expression cassette according to claim 4, wherein said expressioncassette further comprises a transcriptional terminator at a 3′ end ofsaid transgene.
 6. The expression cassette according to claim 4, whereinsaid expression cassette further comprises a secretion signal locatedbetween said purified nucleic acid and said transgene.
 7. The expressioncassette according to claim 4, wherein said transgene codes for a toxin,a fragment thereof, or a variant thereof.
 8. The expression cassetteaccording to claim 7, wherein said toxin is a pathogenic bacteriumtoxin.
 9. A vector comprising the purified nucleic acid according toclaim
 1. 10. The vector according to claim 9, wherein said vector isfunctional in a bacterium.
 11. The vector according to claim 10, whereinsaid bacterium is a Clostridium bacterium.
 12. The vector according toclaim 10, wherein said bacterium is Clostridium perfringens.
 13. Arecombinant cell comprising the purified nucleic acid according toclaim
 1. 14. The recombinant cell according to claim 13, wherein saidrecombinant cell is a prokaryotic cell.
 15. A method for producing apolypeptide, comprising: (a) introducing a transgene coding for saidpolypeptide into a cell, wherein said transgene is under the control ofthe purified nucleic acid according to claim
 1. (b) expressing saidtransgene; and (c) recovering said polypeptide.
 16. A method forproducing a polypeptide, comprising: (a) introducing a transgene codingfor said polypeptide into the recombinant cell according to claim 13,wherein said transgene is placed under the control of said purifiednucleic acid; (b) culturing said recombinant cell to express saidtransgene; and (c) recovering said polypeptide.
 17. The method accordingto claim 15, wherein said cell is a Clostridium bacterium.
 18. Themethod according to claim 15, wherein said polypeptide is a toxin, atoxoid, or a fragment thereof.
 19. A method for producing a polypeptide,wherein said method comprises: (a) introducing the expression cassetteaccording to claim 4 into a cell, wherein said transgene is placed underthe control of said purified nucleic acid; (b) expressing saidtransgene; and (c) recovering said polypeptide.
 20. The vector accordingto claim 9, which further comprises a transgene operably linked to saidpurified nucleic acid.
 21. A recombinant cell comprising the expressioncassette according to claim
 4. 22. A recombinant cell comprising thevector according to claim
 9. 23. A recombinant cell comprising thevector according to claim
 20. 24. The recombinant cell according toclaim 13, wherein said recombinant cell is a bacterium.
 25. Therecombinant cell according to claim 21, wherein said recombinant cell isa bacterium.
 26. The recombinant cell according to claim 22, whereinsaid recombinant cell is a bacterium.
 27. The recombinant cell accordingto claim 23, wherein said recombinant cell is a bacterium.
 28. Themethod according to claim 16, wherein said recombinant cell is aClostridium bacterium.
 29. A method for producing a polypeptide,comprising: (a) culturing the recombinant cell according to claim 21 toexpress said transgene in said expression cassette; and (b) recoveringsaid polypeptide.
 30. A method for producing a polypeptide, comprising:(a) introducing a transgene coding for said polypeptide into therecombinant cell according to claim 22, wherein said transgene is placedunder the control of said purified nucleic acid in said vector; (b)culturing said recombinant cell to express said transgene; and (c)recovering said polypeptide.
 31. A method for producing a polypeptide,wherein said method comprises: (a) culturing the recombinant cellaccording to claim 23 to express said transgene in said vector; and (b)recovering said polypeptide.
 32. A purified nucleic acid comprising SEQID NO:4.
 33. A vector comprising the purified nucleic acid according toclaim
 32. 34. A recombinant cell comprising the purified nucleic acidaccording to claim
 32. 35. An expression cassette comprising a transgeneto be expressed operably linked to the purified nucleic acid accordingto claim
 32. 36. A recombinant cell comprising the expression cassetteaccording to claim
 35. 37. A method of producing a polypeptide,comprising introducing the expression cassette of claim 35 into a cell,culturing the cell to express the transgene; and recovering thepolypeptide.
 38. A purified nucleic acid comprising a sequence from aClostridium strain which hybridizes over the full length of thecomplementary strand of SEQ ID NO:4 under stringent conditions whichcomprise hybridizing at 42° C. in 50% formamide at 5×SSC and1×Denhardt's and which encodes a peptide comprising a hydrophobic regionbordered by charged amino acids that functions as a secretion signalpeptide.